The encapsulation of micro bacteria using a polyvinyl alcohol carrier or a polyurethane carrier is known in the field. The method for the production of a biocatalyst with biologically active material in the form of microorganisms, enzymes, spores and/or cells, which are placed in polyvinyl alcohol gel (referred to as “PVA-gel” in the further text) is disclosed in CZ patent 249 179. PVA-gel as a gel carrier for biologically active material is highly suitable for the production of chemical or biological catalysts. The gel body manufactured according to this method proves higher mechanical stability compared to gel bodies formerly known, particularly in terms of abrasion resistance and tensile strength. Owing to the above mentioned improved mechanical properties, the production of a gel body in a reactive and kinetically suitable lens-shaped form is possible. So the gel body produced is firm and abrasion resistant for more than several months, even at high revolution stirring, compared to those formerly known. Due to the lenticular shape characterized by a large diameter and low height, physically, chemically or biologically active material is always situated closely beneath the surface, which provides its reactive and kinetically useful arrangement. The biotechnological procedures, which utilize PVA-gel as a carrier of biologically active material, are known in the field.
The utilizations of the above mentioned carriers in the process of removing of nitrogen from waste waters are known. Such utilizations are described, for example, in EP0758680 (porous cellulose derivatives), WO09508513 (polyvinyl alcohol, vermiculite, polyurethane) and CN1076488 (polyvinyl alcohol). The mentioned carriers are used (usually) in the immobilization of activated sludge, which is often previously enriched in various ways, in particular by nitrifying bacteria (see, for example, the processes PEGASUS or PEGAZUR), but also directly in the immobilization of pure nitrifying or denitrifying bacteria. As referred to in literature, a very important characteristic of a carrier is the extent of its specific surface with regard to the volume of a reactor, or more precisely to the volume of currently-processed waste waters. That is to say, this parameter is directly related to the size of a reactor and to the waste water holding time in the reactor, because it can be influenced in particular by the amount (or more precisely, by weight or volume) of a carrier with the given specific surface which is placed into a reactor. For example, the nitrogen pollution removal process by means of bacterium immobilized in a polyethylene glycol (PEG) carrier in the shape of tablets with geometrical dimensions S/V=3 and 2, makes it possible to achieve the nitrogen removal rate from 0.34 kg/m3 to 1.14 kg/m3 per day with the content of 2% w/v wet biomass of activated sludge in a carrier. (T. Sumino, H. Nakamura, N. Mori, Y. Kawaguchi: Immobilization of Nitrifying Bacteria by Polyethylene Glycol Prepolymer, Journal of Fermentation and Bioengineering, 73(1), 37-42, 1992.)
However, the extent of the specific (geometric) surface of a carrier with regard to its volume, or more precisely, to its shape and measurements, is coessential parameter, particularly on account of economic reasons. The carriers, shape of which approximates either a ball or cylinder, where the bottom diameter (d) and the height (h) are of an approximately the same value, are still used for removing nitrogen from sewerage water by immobilized cells. The minimal value of the carrier measurement for removing nitrogen from waste waters is 1 mm. The carriers usually used measure 3 mm, which means that the ratio of the surface to the volume of the carrier (S/V) assumes values of approximately 2 mm−1 to 6 mm−1. If a lens-shaped or band-shaped carrier is used, less of a biocatalyst can be used compared to the current processes using the immobilized cells for nitrogen compounds removal from potable, industrial and waste waters, even at the same or higher intensity of the process.
Another alternative use of a gel carrier in biotechnology processes is in the method for lactic acid production. This acid is widely used, particularly in the food, pharmaceutical and chemical industry, amongst others. Lactic acid is produced by a large number of microorganisms, but primarily by filamentous fungi and bacteria. Of the filamentous fungi, Rhizopsus arrhizuz, or oryzae is a good lactic acid producer. The indisputable advantage of it, compared to bacteria, is the fact, that it produces just acid L(+). However, the fermentation mode is aerobic (high sterile air preparation requirements). The aerobic mode of filamentous fungi can be an advantage in the event of direct lactic acid calcium salts preparation, during which prolonging of fermentation and decreasing of yields can occur as a result of inefficient neutralization (weak stirring) in the case of calcium carbonate (Mattey M.: Critical Reviews in Biotechnology 12, 87-132, 1992).
The advantage of bacterial fermentation is its anaerobic mode, hence the simplicity of cultivating devices and lower sterility requirements. Bacteria of the genus Lactobacillus, which convert a sucrose substrate (glucose, sucrose, lactose) at the temperature of 30° C. to 40° C. into lactic acid with different L(+)-, D(−)- and DL acid form content according to properties of the producer, are primarily used for industrial production.
The immobilization of microorganisms by encapsulation into gel carriers is one of the methods, which provide the biomass concentrating in a reactor without a biomass growth in a thoroughly fermented medium. This method consists of enclosing cells into capsules of natural or synthetic gels. The cells must be bigger than the pores of the carrier so that cell release does not occur, while simultaneously ensuring the free diffusion of a substrate and products to the encapsulated cells.
Immobilization of lactic bacteria into different carriers is currently known. Above all, these are carageenans, pectates, alginates of the natural gels (Norton, S. et al.: Enzyme Microbial Technol 16, 457-466, 1994; Richter, K. et al.: Acta Biotechnol. 11, 229-341, 1991; Yan, J. et al.: Chem. Biochem. Eng. Q: 15 (2), 59-63, 2001) and polyacrylamide of the synthetic carriers (Tuli, A. et al.: Enzyme Microbial Technol. 7, 164-168, 1985). The immobilizates are produced in the form of balls with diameter from 3 mm to 5 mm, usually by dropping the relevant gel into a hardening solution. However, the disadvantage of such methods is that the ball-shaped form of the immobilizates causes diffusion limitations inside the balls. The whole gel volume is not used and as a consequence, the microorganisms only grow just under the surface of the ball.
Another suitable method of immobilization is lactic bacteria encapsulation into a polyvinyl alcohol gel. This has numerous advantages compared to other carriers. First of all it is cheap, nontoxic for microorganisms, hardly biodegradable, has excellent physical-mechanical properties, there are no side effects on microorganisms and it shows long term stability (Pat DE 198 27 552.8). Furthermore, the immobilization of cells by the method according to the above mentioned invention is considerate to the productive microorganism (assures a high viability of the microorganism after immobilization and a high rate of surviving microorganisms after immobilization procedure), because the cross-linking of the matrix occurs in a stream of drying air and thus replaces the currently used procedures of cross-linking by freezing (Pat. DE 43 27 923) or hardening in a system with boric acid (U.S. Pat. No. 5,290,693). Furthermore, the lens-shaped form of immobilizates assures optimal loading of the whole volume of the carrier for the growth of microorganisms and the production of lactic acid. The immobilizates prepared by this method can be used repeatedly in batch, semi continual and continual fermentation operation modes.
It is generally known that yeasts Saccharomyces cerevisiae or other yeast microorganisms, which are able to convert glucose and other saccharides into ethanol during the process of fermentation, are traditionally used for the production of ethanol. This fermentation is the basis of beer and wine production but also the foundation of ethanol production for industrial, food and fuel purposes. Recently, growing interest in technologies using bacteria as producers of ethanol has been noticed. Zymomonas mobilis is one of the bacterial producers of ethanol. It is a gram-negative facultatively anaerobic microorganism. It has several advantages compared to yeasts: faster growth of biomass (but generally lower biomass production), higher specific rates of substrate utilization and product creation, it does not need a monitored oxygen supply and produces less side metabolites. It metabolizes glucose and fructose by the Entner-Doudoroff path, which usually occurs with anaerobic microorganisms. In this metabolic path one mole of glucose is converted into 2 moles of ethanol, 2 moles of CO2 and one mole of ATP. This metabolic path minimizes the amount of glucose changed into biomass and in that way increases the ethanol production. The real yield (g alcohol/g glucose) is raised to 0.49 with bacteria, and is usually 0.44 with yeasts).
Fermentation with the assistance of free Zymomonas mobilis cells can occur in batch, semi continual and also continual operation modes (Rogers P. L., Tribe D. E.: U.S. Pat. No. 4,403,034; Rogers P. L., et al., U.S. Pat. No. 4,443,543; Salzbrunn W., et al., U.S. Pat. No. 4,876,196; Bu'lock J. D., Pat GB 2075053).
To date, research interest has focused on the increase of microorganism productivity and on modifying the arrangement of fermentation devices with various innovations; for example cell recycling, the use of flocculating microorganisms and etc. (Arcuri E. J., et al., U.S. Pat. No. 4,413,058; Rogers P. L., et al., U.S. Pat. No. 4,443,544).
The fermentation product (ethanol) is accumulated in a liquid medium and is isolated almost entirely by distillation. It is desirable during product isolation to reduce considerably the content of biomass by sedimentation, centrifuging, filtration and the like, without regard to the arrangement and the technology used. Elevated insoluble content increases distillation energy requirements, causes technological problems (fouling of the distilling apparatus, foaming etc), increases maintenance requirements and can also influence product quality. On the other hand, maximum efficiency of ethanol production must be achieved. The ethanol production rate is directly proportional to the amount of biomass in a fermentor. It means that with constant conditions, increased biomass content causes shortening of the time needed for production of a given amount of ethanol. One of the methods, enabling biomass concentration in a reactor without biomass growth in a thoroughly fermented medium, is microorganism immobilization by encapsulation in gel carriers. This method is based on cells encapsulation into natural or synthetic gel capsules. The cells size must be bigger than the pores size of a carrier so that cell release did not occur, but simultaneously free diffusion of a substrate and products to the encapsulated cells was ensured.
Immobilization of the bacteria genus Zymomonas in various carriers is currently known. Above all, these are carageenans and alginates of the natural gels (Cheetham P. S. J., U.S. Pat. No. 4,393,136; Chibata I., U.S. Pat. No. 4,350,765; Kikuta M., U.S. Pat. No. 5,990,191; Yamada T., et al., U.S. Pat. No. 4,680,263) and polyacrylamide of the synthetic carriers (Yamada T., et al., U.S. Pat. No. 4,680,263; Cheetham P. S. J., U.S. Pat. No. 4,393,136). The immobilizates are usually produced in the form of balls with diameter from 3 mm to 5 mm, mostly by dropping the relevant gel into a hardening solution. However, the disadvantage of such method is that the ball-shaped form of immobilizates causes diffusion limitations inside the balls. The whole gel volume is not used for cell loading and consequently, the microorganism only grows just beneath the surface of a carrier-ball, and does not load the whole volume of the matrix.
A suitable immobilization method is encapsulation the bacterium Zymomonas mobilis into polyvinyl alcohol gel. This has a number of advantages compared to the other carriers. First of all, it is cheap, nontoxic for microorganism, hardly biodegradable, has excellent physical-mechanical properties, there are no side effects on the microorganism and it shows long term stability (Pat. DE 198 27 552.8). Furthermore, the immobilization of cells by the method according to the above mentioned invention is considerate to the productive microorganism (assures a high viability of the microorganism after immobilization and a high rate of surviving microorganisms after immobilization), because the cross-linking of the matrix occurs in a stream of drying air and thus replaces the currently used procedures of cross-linking by freezing (Pat. DE 43 27 923) or hardening in a system with boric acid (U.S. Pat. No. 5,290,693). Furthermore, the lenticular shape of the immobilizates assures optimum utilization of the whole volume of the carrier for the growth of microorganisms and the production of ethanol. The immobilizates prepared by this method can be used repeatedly in batch, semi continual and continual fermentation modes.
Using ball-shaped form of immobilizates, ethanol production was 77 mgethanol/mlgel/h with the aid of Zymomonas mobilis immobilized into carageenan, with 10% by weight of the glucose medium, in a 500 ml batch reactor filled with 20 ml of immobilizates with an immobilizates diameter of 4 mm and a temperature of 30° C., (Chibata I., U.S. Pat. No. 4,350,765). When the producer was immobilized into sodium alginate, where wet cell weight in the immobilizates comprised 20% ww/v (ww—wet cell weight, corresponds to ⅕ of dry cell weight), the productivity under the same conditions was 0.49 gethanol/gww/h in the batch system, which after recalculation corresponds to 98 mgethanol/mlgel/h, and in the continual mode 0.47 gethanol/gww/h, which after digit recalculation corresponds to 94 mgethanol/mlgel/h (Cheetham P. S. J., U.S. Pat. No. 4,393,136).
An alternative use of a gel carrier in biotechnology processes is in the method of producing glucose and fructose from sucrose by the help of immobilized enzyme invertase. Sucrose is a storage disaccharide present mainly in sugar beet and sugar cane. It is used in the food industry and also as a substrate in fermentation processes. It is composed of the monosaccharide glucose and fructose. Sucrose provides these monosaccharides by means of hydrolysis, for example by enzymatic hydrolysis using the enzyme invertase. Its use results in the creation of a mixture of glucose and fructose (invert sugar) which, compared to non-hydrolyzed sucrose, brings a number of advantages: decreased production of crystal turbidity, increased sweetness and usability in fermentation processes.
In industrial practice, enzyme preparations of invertase, which are not recyclable, are used for sucrose hydrolysis. That is the reason why the enzyme becomes one of the biggest economic items in the production of glucose-fructose sirups. Suitable immobilization method of this enzyme provides multiple use of it, and furthermore, it is possible to make the whole production process continuous and by that remarkably increase its effectiveness. The most frequently used technique is cross-linking. The most commonly used cross-linking agents are polyamide (Sou M. et al. JP58086085, Rohrbach R. P. et al. U.S. Pat. No. 426,842), glutaraldehyde (Lee D. M et al. U.S. Pat. No. 4,749,653), polymers with uncombined carboxyl groups (Mauz O. U.S. Pat. No. 4,767,620), polymers with epoxide groups (Mauz O. U.S. Pat. No. 4,931,476), immobilization into foto cross-linking bitumens (Kunihiro I. JP55023941) and embedment on various matrixes by the help of a cross-linking agent, such as immobilization on glass balls by the help of glutaraldehyde. (Toshiyuki Y. et al. JP58179494). Other methods are the covalent attachment of invertase on silica gel particles by the help of glutaraldehyde (Thibauit P. A. EP 0231668), covalent bonding by the help of disulphide bonds on organic and inorganic carriers (Cormier R. A. et al. U.S. Pat. No. 4,176,006), covalent binding by the help of organosilane (Ho G. H. et al. U.S. Pat. No. 4,384,045), covalent binding by the help of dialdehyde on cellulose and lignine materials (Monsan P. U.S. Pat. No. 4,405,715) and covalent binding on dialkylaminoalkyl cellulose (Lapins Ch. D. et al. U.S. Pat. No. 4,933,284). The invertase was also immobilized by adsorption; for example on a matrix from animal bones (Findlay Ch. J. U.S. Pat. No. 5,037,749). Another method is encapsulating cell lysates with high invertase activity into alginate gel (Obana H. et al. JP 57163484, Chang H. N. et al. U.S. Pat. No. 5,766,907) or into polyurethane polymers (Hartdegen F. J. et al. U.S. Pat. No. 4,098,645). Combined techniques, such as for example adsorption of an enzyme on cotton treated by polyethylenimine and subsequent cross-linking by glutaraldehyde (Yamazaki H. et al. CA1203187) also have practical usage.
The use of PVA (polyvinyl alcohol gel) appears to be a very good alternative. The invertase was successfully immobilized into a carrier with a solid center covered by PVA gel (Yamamoto H. and koet al. JP2005042037). Foto cross-linking PVA mixed with polyethylene glycol was also used for covalent binding and invertase enclosing (Ichimura K. JP 58152483, Suehiro T. et al. JP 1252285, Izumida H. et al. JP 1071491). A PVA matrix can also be cross-linked by boric acid, whereas the invertase is encapsulated into the gel (Guocheng Ch. et al. CN 1076488), or by drying (Ishimura F et al. U.S. Pat. No. 4,727,030). Other methods have also been used combined with cross-linking (Tsutsumi S. et al. JP 2046288), or with the addition of vinyl acetate (Moriya T. et al., JP 56113290), vinyl amine (Moriya T. et al., JP 56113292), or aminoacetalized PVA (Yamauchi A. et al. U.S. Pat. No. 4,307,151).
An alternative use of a gel carrier in biotechnological processes is in the method of glucose production from starch by the help of immobilized glucoamylase enzyme. Starch, the nutritive storage matter of plants, is one of the main sources of glucose in food industry and also in the fermentation industry. Starch comprises two types of molecules: amylase and amylopectin. Amylase, in corn for example, represents 10% of total starch. It comprises up to 1 000 glucose units, linked by α-1,4-glycoside bonds. The remaining 90% represents amylopectin in which, apart from α-1,4-glycoside bonds, α-1,6-glycoside bonds are also included. The number of glucose units in the chain is up to 10 000.
The process of starch raw material pretreatment for usage in fermentation processes consists of two steps. In the first step, starch liquefaction comes about by the help of endoenzyme α-amylase, which randomly dissociates α-1,4-glycoside bonds at a high temperature. This reaction causes the formation of dextrins and lower oligosaccharides with different chain lengths.
In the second step, dextrins and oligosaccharides are dissociated from the non-reducing end by exoenzyme glucoamylase into glucose units. Glucoamylase dissociates both α-1,4-, and α-1,6-glycoside bonds. However, α-1,6-glycoside bonds are hydrolyzed at a much lower rate. The rate of the hydrolysis also depends on the chain length.
In industrial practice, from 1 l to 1.2 l of glucoamylase enzyme preparation, which is not recyclable, is used per tonne of starch. Suitable immobilization of that enzyme would allow its multiple use, with the possibility of making the whole process continual. Significant improvement has been achieved with immobilization of this enzyme by cross-linking with an agent, such as polyamine (Symon et al. U.S. Pat. No. 4,415,663, Lantero et al. U.S. Pat. No. 5,472,861, DeFilippi U.S. Pat. No. 4,343,901, Rohrbach et al. U.S. Pat. No. 4,268,423), glutaraldehyde (Lee et al. U.S. Pat. No. 4,749,653, Nishimura et al. U.S. Pat. No. 4,888,285, Rorvah et al. K.R. 8601229), acryl or alyl agents (Boross et al. U.S. Pat. No. 4,794,083, Selemenev et al. RU 2204600), by means of which the enzyme is anchored on an inorganic or organic matrix. The usage of adsorption methods (Abdullah et al. U.S. Pat. No. 4,226,937, Kumakura et al. JP 61060700, Motai et al. J.P. 59232092, Selemenev et al. R.U. 2181770, Kimura et al. J.P. 63056297), and also membrane reactors with immobilized glucoamylase (Thomas et al. U.S. Pat. No. 5,130,237) appears to be suitable. Encapsulation, which biological material encapsulates into a gel structure and is used primarily for microorganism immobilization, is entirely absent during glucoamylase immobilization. However, without cross-linking into bigger structures the enzyme is easily washed out from matrix pores.
An alternative use of a gel carrier in biotechnology processes is in the method of lactose solution hydrolysis and the production of D-galactose, D-glucose and galactooligosaccharides from lactose solutions by the help of immobilized β-galactosidase.
Disaccharide lactose is synthesized in the mammary glands of most mammals. It is commercially produced from cows' milk (with a lactose content of 4.5% to 5% w.) by extraction from whey (Baldrick and Bamford, 1997). Lactose present in milk and milk products represents an important nutritional item. It supports the growth of Bifidobacterium sp., it is the source of galactose needed for the production of galactolipides and galactooligosaccharides, it helps in calcium absorption, etc. (Maldonado et al., 1998). It has a low solubility and it crystallizes at a concentration of more than 18% w. That appears to be a problem, because the lactose crystals cause a disagreeable sand-like texture (Zadow, 1992) in the production of dairy products, such as condensed milk and ice-cream. This is the reason why lactose hydrolysis is highly desirable for these products. Furthermore, lactose hydrolysis also causes a considerable decrease in the hygroscopicity of dried milk products, an increase in sweetness and the process of the Maillard reactions is reduced ({hacek over (C)}urda et al., 2001, Zadow, 1992, Rosenberg et al., 1995).
β-galactosidase is not only significant in lactose hydrolysis in milk, but also in whey processing. Whey is produced in quite large amounts as a side product of the dairy industry during the production of cheese, cottage cheese and casein. During cheese production, more than 150 million tons of whey is produced annually in the world (on average, 10 l of whey per kg of cheese) and world-wide cheese production has been increasing constantly. Whey is still not extensively processed, which represents an economic and ecological problem (Novalin et al., 2005). Furthermore, the handling thereof entails many restrictions and it is not possible to freely release it into the environment. Half of the waste product produced is used in the production of whey protein concentrates (WPC), but primarily for feeding farm animals (Rudolfová and {hacek over (C)}urda, 2005). Whey can be also used as a raw material for ethanol production. In consideration of this fact, it could be an attractive substrate for fermentation processes in the future (Coté et al., 2004). For example, during ethanol production it is possible to use Kluyveromyces yeast which utilizes lactose. Lactose hydrolysis enables the use of thickened whey with a higher concentration of C-source as a substrate and thereby considerably increases the fermentation yield (Rosenberg et al., 1995). In some cases it is possible to perform the fermentation process in such a way that the productive microorganism utilizes only the glucose present, while the remaining galactose can be isolated, purified or chemically modified afterwards (Rosenberg, 2000).
Another significant use of β-galactosidase in the food industry is the creation of galactooligosaccharides (GOS). They are simultaneously formed during lactose hydrolysis due to the transglycozylate activity of β-galactosidase. β-galactosidase transglycozylate activity was first described in the 1950's (Aronson, 1952). The first published works focused on monitoring the favorable effects of GOS and the search for optimal methods of their production (Mahoney, 1998).
GOS belongs to the group of so-called probiotics: indigestible food components which selectively stimulate the growth or, more precisely, the activity of probiotic cultures in the human digestive tract. Owing to their β-configuration, GOS are resistant to hydrolysis by saliva and digestive fluid enzymes, which hydrolysis β-glycoside bonds selectively (Sako et al., 1999). GOS pass into the large intestine, where they participate in many important processes. Bifidogenous microflora metabolizes GOS into short chain fatty acids (acetic, propionic, lactic and butyric acid) and into gases (Johnson et al., 1993). Incipient acids stimulate peristalsis of the intestine and aid the absorption of calcium and iron by decreasing pH. GOS are also known as bifidobacterium growth factors, which are recognized for their beneficial health effects. Furthermore, bifidobacteria selectively utilizes galactooligosaccharides, which suppresses the growth of undesirable microorganisms in the digestive tract (Pennisin, 1997). GOS are also beneficial for oral cavity health, when not utilized by oral micro flora (Streptococcus mutants), and thus prevent the formation of dental caries (Szilagyi, 1999).
The individual β-galactosidases differ in the total amount and structure of GOS produced. For example, after isolating β-galactosidases from different strains of Bifidobacterium genus and their subsequent use during GOS synthesis from a 30% lactose solution, there was an evident difference in the amount produced. In the case of Bifidobacterium angulatum, they formed 43.8% of all saccharides present in a solution, whilst in the case of Bifidobacterium pseudolongum it was only 26.8% (Rabiu et al., 2001). Regarding the structure, the type of the newly formed glycoside bond of two monosaccharide units (galactose-galactose, galactose-glucose) is in the case of Bifidobacterium bifidum β(1-3) (Dumortier et al., 1994), as opposed to β(1-4) in Bacillus circulans (Mozaffar et al., 1984) and β(1-6) in Streptococcus thermophilus (Matsumoto, 1990).
Enzyme, β-galactosidase was immobilized in the following ways: entrapment (Mammarella and Rubiolo, 2005, Rodriguez-Nogales and Delgadillo, 2005), cross-linking (Sungur and Akbulut, 1994), adsorption (Carpio et al., 2000), covalent binding (Hu et al., 1993, Findlay, 1991, Di Serio et al., 2003), enclosure in membranes (Novalin et al., 2005), or a combination of these methods (more closely described in part 1.2.1).
The β-galactosidase immobilization process is associated with certain disadvantages, such as the loss of enzyme activity after immobilization. The decrease of β-galactosidase activity after immobilization ranges from 5% to 90% and depends on the immobilization method used. This disadvantage is compensated by the possibility of reusability of an immobilizates. The ability to preserve an enzymes activity and the stability of an immobilizates during repeated usage are the decisive parameters for the application of an immobilizates on an industrial scale (Tanaka and Kawamoto, 1999).
Polyacrylamide and a polyvinyl alcohol gel are often used for the method of entrapment. A PVA matrix was also used for encapsulation of β-galactosidase isolated from filamentous fungi, whereby its temperature stability was increased. The enzyme retained 70% of its previous activity after 24 hours at 50° C. and 5% of its previous activity at 60° C. (Batsalova et al., 1987). Khare and Gupta (1988) immobilized β-galactosidase from E. coli using a combination of the two methods: cross-linking and subsequent encapsulation into a polyacrylamide gel. In the same procedure, but using dimethyladipimidate as a cross-linking agent and enzyme preventing substances: bovine serum albumin, cysteine and lactose, the activity measured 190% of the previous immobilizer activity. The comparison of different methods of β-galactosidase immobilization from thermophilic bacterium Thermus aquaticus, showed that cross-linking and subsequent entrapment into agarose granula is the preferable process for the immobilization of high concentrations of enzyme with the benefit of high enzyme activity (Berger et al., 1995). The process of immobilization enables increased enzyme stability under different reaction conditions. The entrapment of β-galactosidase from A. oryzae into porous PVA cryogel increased the temperature resistance of the enzyme, pH value and ionic strength (Rossi et al., 1999).
Adsorption is a technically undemanding method of immobilization. Hydrophobic cotton fiber can serve, for example, as a carrier on which an enzyme preserves 50% of its previous activity (Sharma and Yamazaki, 1984). Bakken et al., used β-galactosidase from A. oryzae by adsorbing it on polyvinylchloride and silica gel in the form of a membrane (1990) during lactose hydrolysis in milk in an axial flow reactor. β-galactosidase immobilized by adsorption on bone powder preserved 83% of its previous activity, but during four batch hydrolyses the immobilizates gradually lost its activity. After the fourth hydrolysis, the activity of the immobilizates was just 24% of the original (Carpio et al., 2000). This experiment showed the disadvantage of the immobilization method, during which slight desorption occurs. This effect can be eliminated by the addition of a suitable cross-linking agent (Szczodrak, 2000).
Piettaa et al. (1989) immobilized β-galactosidase isolated from A. oryzae into two different carriers using a covalent bond. The first was zeolite, but it was not convenient because it only binds a small amount of enzyme. Contrary to that, powder nylon-6 covalently bound a larger amount of the enzyme and created a stable complex. Peters and Rehm (2006) immobilized β-galactosidase by covalent binding onto polyhydroxyalkanoate granules. The immobilizates gained was stable in the long term during its storage under different conditions, which provides evidence of a strong bond between the carrier and the immobilized enzyme.
Glutardialdehyde with two reactive functional groups is the most frequently used cross-linking agent in β-galactosidase immobilization (Guisán et al., 1997, Szczodrak, 2000, Zhou and Chen 2, 2001). However, the method of cross-linking immobilization is particularly used in combination with other methods (above mentioned, Panesar et al., in press), such as adsorption or entrapment. For example β-galactosidase from A. oryzae immobilized using the entrapment method into the form of fibers composed from alginate and gelatine, was then cross-linked by glutardialdehyde, which prevented washing out of the enzyme (Tanriseven and Dogan, 2002). This type of cross-linking was also used in β-galactosidase immobilization into a cobalt-alginate gel. However, cobalt release during lactose hydrolysis made the use of the immobilizates impossible in the food industry (Ates and Mehmetoglu, 1997).
The activity and stability of the immobilized enzyme is affected by pH value, temperature and ionic strength (Roy and Gupta, 2003). Compared to a free enzyme, the immobilized enzyme has wider optimum pH and temperature ranges as mentioned in most published works. The higher stability of immobilized β-galactosidase at a lower pH value and higher temperature is an advantage in lactose hydrolysis, and furthermore, by decreasing the pH value and increasing the temperature the risk of contamination is reduced. On the other hand, the shift of the pH optimum and the stability increase is, under these conditions, an advantage during lactose hydrolysis in sweet whey, whose pH is within the 5.5 to 6.0 interval (Szczodrak, 2000).
β-galactosidase from different microbial sources was also immobilized for the purpose of production galactooligosaccharides in the continuous as well as in the batch operation mode (Chockchaisawasdee et al. 2005). Compared to a free enzyme, the immobilized enzyme produced lower GOS concentrations. This decrease was caused by the limitation of substrate access to the enzyme in the immobilized system (Mahoney, 1998). The concentration and structure of emerging GOS depended to considerable extent on the initial substrate concentration and on the origin of the enzyme. The increase in the initial lactose concentration from 14% to 23% doubled the GOS production (Foda and Lopez-Leiva, 2000, Chockchaisawasdee et al., 2005).
Shin et al. (1998) immobilized an enzyme isolated from Bullera singularis ATTC 24193 into chitozane granules. They used the thus immobilized enzyme in GOS production. The experiment lasted for 15 days, during which time they registered 55% w. of GOS of all saccharides (the initial substrate concentration being 100 g·l−1) and volume productivity of 4.4 g·l−1·h−1. Another example of immobilized β-galactosidase usage in oligosaccharides production was also described in the work of Albayrak and Yang (2002). They reached the maximum production 26% of weight of all saccharides (with a substrate concentration 400 g·l−1) and the volume productivity 106 g·l−1·h−1 by using an enzyme from A. oryzae immobilized on cotton fiber. Foda and Lopez-Leiva (2000) tested a membrane flow reactor with immobilized β-galactosidase for GOS preparation. They reached the highest production (31% of weight) by using whey treated by ultrafiltration with an initial lactose concentration of 20% w. Whey is a rich source of lactose; its usage in GOS production appears to be very attractive.
There is a known casting unit which is used in the production of immobilizates, which are products—biocatalysts on the basis of polyvinyl alcohol carrier—usually manufactured in the lens-shaped form or small belts. The mixture of polyvinyl alcohol gel and biomass is applied to a continuous conveyor belt using a dropping, casting mechanism in accurate and previously defined dosages, where it is changed into an adhesive mass by drying and physically gelation during the production process. At present, the casting unit for the production of immobilizates based on a polyvinyl alcohol carrier consists of a casting mechanism arranged in front of the drying channel, inside which a continuous conveyor belt (made from polyethylene, for example) runs. The casting mechanism, known as a casting head, is fitted with one row of casting needle injectors with a diameter varying from 0.1 mm to 2.00 mm, which pulsate by the help of electromagnets with different frequency and different pulse length and transfer or pour the mixture of polyvinyl alcohol gel and biomass on the continuous conveyor belt. The casting unit further contains a reswelling section placed in the upper part of the drying channel, which is formed by a system of low-pressure spraying jets, which wets (reswell) the dried product. Furthermore, the casting unit is equipped with a collecting part, comprising of a pressure wiper made from stainless steel plate rinsed by spraying jets placed above the pressure stainless steel wiper and mounted on the carrying frame of the casting unit, above the drive cylinder of the belt. Ambient or dehumidified air are used as the source of drying media using continuous atmospheric dehumidification or continual freezing, which is warmed up by heating elements before entering the drying channel, and which ensures gelation by drying the immobilizates produced on the basis of a polyvinyl alcohol carrier. Casting units arranged in this manner are incapable of long-term economic production, in particular due to the difficulty in removing the immobilizates on the base of the polyvinyl alcohol carrier from the conveyor belt. A mechanical stainless steel wiper is used for wiping, and following collection. The wiper is firmly pressed against the surface of the conveyor belt, i.e. on the basis of elasticity, and subsequently, the mass is wiped off by the wiper and falls off into a collecting reservoir. This mechanical removal of the product from the belt is far from perfect, particularly considering the formation of big clusters, agglomerates of polyvinyl alcohol carrier, and the consequent production of a product with low or zero activity. A further disadvantage is also considerable mechanical wear of the casting unit's continuous conveyor belt. It was further learned, that the use of salt solutions in the reswelling part of the casting unit causes salts to cling to the surface of the continuous belt and consequently changes to the surface tension of the casting unit's the conveyor belt, hence the change in the shape of the product. This change means that during the removal and unsticking of the immobilizates from the conveyor belt damage and the formation of large clusters or agglomerates is unavoidable in most cases.